During infections by pathogens, such as viruses, bacteria, and parasites that invade human cells, an important component of the immune defense against infection are natural killer (NK) cells. NK cells are a group of lymphocytes that differ from B and T lymphocytes by responding much faster to infections. Unlike B and T cells, NK cells do not express antigen-specific receptors. Therefore, proliferation and activation of NK cells does not begin with a few rare NK clones but with a large number of NK cells. Various chemokines and cytokines, such as type I interferons or interleukin (IL)-12, which are produced early during infections, activate NK cell responses. NK cells exert their function in two ways: by producing cytokines, such as interferon-gamma, and by killing infected cells. NK cells can also kill tumor cells. Despite the importance of NK cells in the innate response to many types of pathogens, it is still unclear what receptors and what signal transduction pathways control their activation. NK cell inhibitory receptors that recognize surface molecules called major histocompatibility complex class I (MHC class I) prevent the killing of normal, healthy cells. The major goal of this project is to define the receptor-ligand interactions that regulate cytotoxicity by NK cells. Cytotoxicity of human NK cells is activated by receptors that bind ligands on target cells, but the relative contribution of the many different activating and inhibitory NK cell receptors is difficult to assess. Here we describe an experimental system that circumvents some of the difficulties. Adhesion through beta2 integrin LFA-1 is a common requirement of cytotoxic T cells and NK cells for efficient lysis of target cells. However, the contribution of LFA-1 to activation signals for NK cell cytotoxicity, besides its role in adhesion, is unclear. The role of LFA-1 was evaluated by exposing NK cells to human ICAM-1 (a ligand of LFA-1) that was either expressed on a Drosophila insect cell line, or directly coupled to beads. Expression of ICAM-1 on insect cells was sufficient to induce lysis by NK cells through LFA-1. Co-expression of peptide-loaded HLA-C with ICAM-1 on insect cells blocked the LFA-1-dependent cytotoxicity of NK cells that expressed HLA-C-specific inhibitory receptors. Polarization of cytotoxic granules in NK cells towards ICAM-1 and ICAM-2-coated beads showed that engagement of LFA-1 alone is sufficient to initiate activation signals in NK cells. Thus, in contrast to T cells, in which even adhesion through LFA-1 is dependent on signals from other receptors, NK cells receive early activation signals directly through LFA-1. The guanine nucleotide exchange factor Vav1 regulates actin polymerization and contributes to cytotoxicity by natural killer (NK) cells. An open question is how Vav1 becomes activated and what receptor can signal upstream of actin cytoskeleton rearrangement upon NK cell contact with target cells. Using transfected insect cells that express ligands of human NK cell receptors, we show that engagement of the beta2 integrin LFA-1 on NK cells by ICAM-1 led to a tyrosine phosphorylation of Vav1 that was not sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 phosphorylation was blocked by an inhibitor of src-family kinases, and correlated with activation of its downstream effector PAK. Binding of activation receptor 2B4 to its ligand CD48 was not sufficient for Vav1 phosphorylation. However, co-engagement of 2B4 with LFA-1 resulted in an enhancement of Vav1 phosphorylation that was sensitive to cholesterol depletion and to inhibition of actin polymerization. Vav1 was recruited to cholesterol-enriched lipid rafts only when 2B4 and LFA-1 were co-engaged, but not after LFA-1 engagement. Therefore, binding of LFA-1 to ICAM-1 on target cells may initiate an early signaling cascade in NK cells through activation of Vav1, leading to cytoskeleton reorganization and amplification of signals from other activation receptors.